Human papillomavirus (HPV) is a small size virus with cyclic double stranded DNA of about 8000 base pairs in a genome, and causes human specific infection. More than 100 kinds of HPV subtypes have been reported up to present, which are largely divided into those detected in skin lesions such as wart and the like (skin tropic HPV), and those detected in mucous membrane lesions (mucosa tropic HPV). Moreover, mucosa tropic HPV is divided into a high-risk type (HPV16, HPV18 etc.) involved in cancers such as cervical cancer, perianal cancer, penile cancer and the like, and a low-risk type (HPV6, HPV11 etc.) involved in benign condyloma acuminatum and the like. WHO estimates that HPV infection is involved in 11% of malignant tumors of women around the world or about 450,000 people, and there are 300 million HPV infection carriers in the world, suggesting that HPV is an ordinary virus. Particularly, HPV is almost always detected in cervical cancer patients.
Cervical cancer is the second highest in number among the cancers of women in the world, and about 250,000 people in the world, and about 2500 people in Japan die of cervical cancer annually. Thus, the prophylaxis, diagnosis, and treatment of cervical cancer have been recognized as highly important issues for the women's health. Fortunately, prophylaxis of cervical cancer is possible. The reasons therefor are that dysplasia, which is a precancerous lesion of cervical cancer, can be found by cytological diagnosis and the like, and dysplasia can be cured by a simple treatment. Since dysplasia is caused by HPV infection, the importance of HPV genetic screening, which enables earlier and more certain discovery of cervical cancer or precancerous lesion, has been increasingly recognized.
The method of HPV genetic screening is largely divided into a method including direct detection of HPV DNA, and a method including amplification of HPV DNA by polymerase chain reaction (PCR) before detection of the DNA.
A representative method without using PCR is a hybrid capture method. In this method, RNA probe is hybridized with HPV DNA in a sample, and the resulting DNA/RNA hybrid is detected by immunoassay using an antibody. Being convenient, this method is currently a main method for HPV genetic screening. On the other hand, the method has low reliability due to low sensitivity, low stability caused by the use of RNA probe, and uneliminatable possibility of contamination. Moreover, even though this method can be used to identify whether the infected HPV is of a high-risk type or a low-risk type, it cannot identify its subtype.
In a method utilizing PCR, amplified DNA is detected by a method including cleavage with a restriction enzyme and electrophoresis to detect bands, or a method including hybridization with a probe on DNA microarray for detection. The former detection method utilizing electrophoresis can be performed in a general laboratory facility and is superior in the economic aspect. As defects, however, it requires complicated processes and incompletely identifies subtypes. On the other hand, the latter detection method utilizing hybridization with a probe shows high detection sensitivity and high specificity of subtype identification. However, it has problems in the rapidness and the economic aspect.
As for DNA amplification by PCR, a method of type-specific amplification of HPV gene using a type-specific primer, and a method of non-type-specific amplification of HPV gene using a random primer or non-type-specific primer are known.
In the methods disclosed in patent documents 1 and 2, DNA is amplified by PCR using a type-specific primer. The amplified DNA is subjected to hybridization with DNA microarray in patent document 1 using an amplified double stranded DNA per se as a probe, and subjected to electrophoresis in patent document 2 to detect presence or absence of HPV DNA. These methods achieve amplification with high specificity and high sensitivity by the use of a type-specific primer. Even when a mixture of primers is used, they can specifically detect a particular type of HPV.
On the other hand, in the method disclosed in patent document 3, HPV DNA is amplified using a non-type-specific primer, and hybridized with DNA microarray using a short single strand oligo DNA as a probe to detect HPV DNA. Since this method uses a common primer, it is advantageous in the synthesis cost and easiness of design. However, overall sensitivity is low even if low specificity and low sensitivity in PCR are overcome to a certain degree by increasing the specificity of hybridization by the use of a short probe. Even though a primer is prepared for each HPV type in the method disclosed in patent document 4, since the respective primer sequences are almost the same, the method of preparing the primers is considered to be almost the same as in patent document 3.
As the situation stands, a method capable of detecting multiple types of HPV with high specificity and high sensitivity, which is superior in rapidness, convenience and economic aspects needs to be developed.                [patent document 1] JP-A-2004-121240        [patent document 2] JP-A-2006-345800        [patent document 3] Patent Application Publication No. 2005-503177        [patent document 4] Patent Application Publication No. 2005-519611        